Mucin secreted into the alimentary tract often forms a mucus layer on the mucosa and is believed to protect the underlying epithelium against various factors in the lumen. We developed an improved histological technique for the evaluation of the mucus layer in the rat cecum. We used this technique to compare the effect of three nonstarch poly- and oligosaccharides on the status of mucus layer. Rats were divided into four groups (fiber-free [FF], cellulose [CEL], fructooligosaccharide [FOS], or guar gum [GG]). The frozen cecum with its contents was cut into cross-sections (5 mm thick) and fixed overnight in half-strength Bouin's solution. The sections were then transferred to 80% ethanol for 24 h. After being stained with alcian green, the mucus layers were clearly visualized in thin sections of the rat cecum, except for those that received FOS where the mucus layer had disappeared; the strong signal of mucus was seen in the cecal digesta of FOS-fed rats. Our histological method successfully provided information about the status of mucus layer that is important for an assessment of the epithelial state in the intestine.